Antitumor antibodies, proteins, and uses thereof

ABSTRACT

Antibodies that bind to a 40 kDa protein which is expressed on tumors, but is not expressed on normal adult hemopoietic cells are disclosed. Also disclosed are methods for production and the use of such antibodies.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.11/491,500, filed Jul. 21, 2006, which is a continuation of U.S.application Ser. No. 10/747,031, filed Dec. 23, 2003, which is acontinuation of U.S. application Ser. No. 09/618,421, filed Jul. 18,2000, now U.S. Pat. No. 6,693,176, which, in turn, claims priority toU.S. Provisional Patent Application Ser. No. 60/145,337, filed Jul. 23,1999. The contents of all of the aforementioned patents and patentapplications are incorporated herein by reference.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under grant numberR01CA55233-06, awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to antibodies and the proteins to whichthey specifically bind, and to methods for production and the use ofsuch antibodies that specifically bind to tumor cells.

BACKGROUND OF THE INVENTION

The E710.2.3 cell line is a cloned murine CD4-CD8-thymic T lymphoma cellline, originally isolated from a thymic tumor of an AKR/J mouse. Whencultured by itself at low density, E710.2.3 does not proliferatespontaneously, unless it is stimulated with phorbol 12-myristate13-acetate (PMA). E710.2.3 can be stimulated to proliferate by contactwith thymocytes or splenocytes. However, E710.2.3 can proliferatespontaneously when cultured at high density in the absence of PMA orother cells. When E710.2.3 is injected into syngeneic mice it grows as amalignant tumor in lymphoid organs and the thymus.

SUMMARY OF THE INVENTION

The invention is based on the discovery of monoclonal antibodies thatcan specifically bind to a 40 kDa protein expressed on the surface ofnumerous types of tumor cells, but do not bind to adult normalhematopoietic cells. The new monoclonal antibodies can blockproliferation and induce apoptosis of tumor cells to which theyspecifically bind.

Based on these discoveries, the invention features monoclonalantibodies, or antigen-binding fragments thereof, wherein the monoclonalantibodies (a) bind to fetal thymocytes, (b) inhibit cell proliferationof a cell upon binding to the cell, and (c) do not bind to adultthymocytes. The monoclonal antibodies can also induce homotypicaggregation upon binding to a cell, induce apoptosis in a cell to whichthey bind, and can specifically bind to one or more tumor cell lines inthe group E710.2.3, RMA-S, CTLL, LB 17.4, A20, WEHI-231, PBK101A2, C2.3,B16, MC57, WOP-3027, 293T, 143Btk, Jurkat, and Cos. The antibodies canbe labeled, e.g., with a detectable label.

Also within the invention is the monoclonal antibody DMF10.62.3 producedby the hybridoma cell line ATCC No. PTA-377, the monoclonal antibody DMF10.167.4 produced by the hybridoma cell line ATCC No. PTA-405, and themonoclonal antibody DMF10.34.36 produced by the hybridoma cell line ATCCNo. PTA-404.

The invention also features monoclonal antibodies, that bind to the sameprotein as the protein bound by the monoclonal antibody produced byhybridoma cell line ATCC No. PTA-377, hybridoma cell line ATCC No.PTA-405, or hybridoma cell line ATCC No. PTA-404. The monoclonalantibody can be humanized.

In another aspect, the invention features monoclonal antibodies, orantigen-binding fragments thereof, that bind specifically to a 40 kDaprotein bound by the monoclonal antibody produced by hybridoma cell lineATCC No. PTA-377, hybridoma cell line ATCC No. PTA-405, or hybridomacell line ATCC No. PTA-404.

In yet another aspect, the invention features chimeric monoclonalantibodies, or antigen-binding fragments thereof, that bind to the sameprotein as the protein bound by the monoclonal antibody produced byhybridoma cell line ATCC No. PTA-377, hybridoma cell line ATCC No.PTA-405, or hybridoma cell line ATCC No. PTA-404, wherein the chimericantibodies include non-human variable regions and human constant regionsof light and heavy chains.

In still another aspect, the invention features monoclonal antibodies,or antigen-binding fragments thereof, that bind to the same epitope asthe epitope bound by the monoclonal antibody produced by hybridoma cellline hybridoma cell line ATCC No. PTA-377, hybridoma cell line ATCC No.PTA-405, or hybridoma cell line ATCC No. PTA-404.

The invention also features monoclonal antibodies, or antigen-bindingfragments thereof, that bind specifically to a protein characterized by(i) a molecular weight of 40 kDa, (ii) expression on the surface offetal thymocytes, (iii) no expression on the surface of adultthymocytes, (iv) the ability to block cell proliferation upon binding bythe antibody, and (v) the ability to induce homotypic aggregation uponbinding by the antibody. The monoclonal antibody binds specifically to aprotein that is further characterized by (vi) the ability to induceapoptosis in a cell upon binding by the antibody, and (vii) expressionon the surface of a group of tumor cell lines consisting of E710.2.3,RMA-S, CTLL, LB 17.4, A20, WEHI-231, PBK101A2, C2.3, B16, MC57,WOP-3027, 293T, 143Btk, Jurkat, and Cos.

The invention further features an antigen-binding fragments of themonoclonal antibodies described herein. The antigen binding fragmentscan be labeled, e.g., with a detectable label.

The invention also features the hybridoma cell lines that produce themonoclonal antibodies described herein. For example, the inventionfeatures the hybridoma cell line ATCC No. PTA-377, hybridoma cell lineATCC No. PTA-405, or hybridoma cell line ATCC No. PTA404.

The invention further features a substantially pure proteincharacterized by (i) a molecular weight of 40 kDa, (ii) expression onthe surface of fetal thymocytes, (iii) no expression on the cell surfaceof adult thymocytes, (iv) the ability to block cell proliferation uponbinding by the antibody described herein, and (v) the ability to inducehomotypic aggregation upon binding by the antibody described herein. Theprotein can be further characterized by (vi) expression on a group oftumor cell lines consisting of E710.2.3, RMA-S, CTLL, LB17.4, A20,WEHI-231, -PBK101A2, C2.3, B16, MC57, WOP-3027, 293T, 143Btk, Jurkat,and Cos, and (vii) the ability to induce apoptosis in a cell uponbinding by the antibody described herein.

In another aspect, the invention features substantially pure proteinsthat bind to the monoclonal antibody produced by hybridoma cell lineATCC No. PTA-377, hybridoma cell line ATCC No. PTA405, or hybridoma cellline ATCC No. PTA404.

The invention also features a pharmaceutical composition comprising themonoclonal antibody described herein and a pharmaceutically acceptablecarrier.

The invention further features a method for detecting a tumor cell in asubject. The method includes contacting a cell sample from the subjectwith one or more of the monoclonal antibodies described herein, anddetecting binding of the antibody to the sample, wherein bindingindicates the presence of a tumor cell in the subject. Examples of tumorcells include thymic lymphoma, T-cell tumor, a B-cell lymphoma,melanoma, osteosarcoma, and acute T-cell leukemia. The tumor cell mayalso be in a patient. The monoclonal antibodies used to detect tumorscan be labelled.

The invention also features a method of inhibiting tumor cellproliferation. The method includes contacting the tumor cell with aquantity of the monoclonal antibodies described herein, sufficient toinhibit proliferation of the tumor cell.

In another embodiment, the invention features a method of inducingapoptosis in a cell. The method includes contacting the cell with aquantity of one or more of the monoclonal antibodies described hereinsufficient to induce apoptosis in the cell. The cell can be a tumor cellselected from the group consisting of thymic lymphoma, T-cell tumor, aB-cell lymphoma, melanoma, osteosarcoma, and acute T-cell leukemia. Thecell can be in vitro or in vivo.

The invention also encompasses a kit for tumor diagnosis, including oneor more of the monoclonal antibodies described herein and instructionsfor its use. The kit can contain a tumor cell selected from the groupconsisting of thymic lymphoma, T cell tumor, a B-cell lymphoma,melanoma, osteosarcoma, and acute T cell leukemia.

The invention also includes a tumor cell targeting agent including oneor more of the monoclonal antibodies described herein, which can beconjugated to a moiety to deliver the moiety to a tumor cell. Examplesof moieties include anti-tumor agents, cytotoxins, cytokines, orreporter groups.

Another embodiment of the invention is a method of selectivelydelivering a moiety to a tumor cell in a mammal. The method includesadministering to the mammal the targeting agent described herein linkedto the moiety and allowing sufficient time for the targeting agent toreach the tumor cell wherein the antibody in the targeting agent bindsto the tumor agent described herein linked to the moiety and allowingsufficient time for the targeting agent to reach the tumor cell whereinthe antibody in the targeting agent binds to the tumor cell, therebyselectively delivering the moiety to the tumor cell in the mammal.Examples of moieties include antitumor agents, cytotoxins, cytokines,and reporter groups.

The invention further encompasses a method of isolating the 40 kDaprotein described herein. The method includes contacting a samplecontaining the protein with the monoclonal antibodies described hereinfor a time and under conditions sufficient to enable the formation ofmonoclonal antibody/protein complexes, removing one or more of thecomplexes, if any, from the sample and removing the protein from thecomplex, thereby isolating the protein.

An “isolated nucleic acid sequence” is a nucleic acid sequence that issubstantially free of the genes that flank the nucleic acid sequence inthe genome of the organism in which it naturally occurs. The termtherefore includes a recombinant nucleic acid sequence incorporated intoa vector, into an autonomously replicating plasmid or virus, or into thegenomic nucleic acid sequence of a prokaryote or eukaryote. It alsoincludes a separate molecule such as a cDNA, a genomic fragment, afragment produced by polymerase chain reaction (PCR), or a restrictionfragment.

An antibody that “specifically binds” to a protein is one that binds toa protein, but which does not recognize and bind to other molecules in asample, e.g., a biological sample, which naturally includes the protein,e.g., the 40 kDa protein.

“Conservative” amino acid substitutions are substitutions in which oneamino acid residue is replaced with another amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Any one of a family of amino acids can be used to replaceany other members of the family in a conservative substitution.

The terms “polypeptide, peptide, and protein” are used interchangeablyherein to refer to a chain of amino acid residues.

An “antigen-binding fragment” of an antibody is a portion of theantibody that is capable of binding to an epitope on an antigen, e.g.,the 40 kDa protein, bound by the full antibody.

An “epitope” is a particular region of an antigen, e.g., a protein towhich an antibody binds and which is capable of eliciting an immuneresponse.

A “substantially pure” 40 kDa protein is a 40 kDa protein that is atleast 60%, by weight, free from the proteins and naturally-occurringorganic molecules with which it is naturally associated. Preferably, thepreparation is at least 75%, more preferably at least 90%, and mostpreferably at least 99%, by weight 40 kDa protein. A substantially pure40 kDa protein can be obtained, for example, by affinity chromatographyusing antibodies or monoclonal antibodies described herein, and/or byphysical purification techniques.

An “isolated” antibody is an antibody which is substantially free fromother naturally-occurring organic molecules with which it is naturallyassociated.

An antibody or other molecule that blocks cell proliferation is anantibody or molecule that inhibits cell cycle, division, or both.

By “homotypic aggregation” is meant a biologically active processwhereby cells of the same type are stimulated to adhere to one another.

A “reporter group” is a molecule or compound that has a physical orchemical characteristic such as luminescence, fluorescence, enzymaticactivity, electron density, or radioactivity that can be readilymeasured or detected by appropriate detector systems or procedures.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

The invention features antibodies that recognize a 40 kDa protein whichis expressed on tumor cells. The antibodies can be used to inhibitproliferation of tumor cells and induce apoptosis of tumor cells towhich they specifically bind. The monoclonal antibodies can be useddiagnostically (e.g., to determine the presence of malignant cells), orcan be used therapeutically to treat tumor cells by themselves orthrough their delivery of an attached antitumor agent.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWING

FIGS. 1A-D are four flow cytometric graphs showing the expression of the40 kDa protein on E710.2.3 (FIG. 1A), A20 (FIG. 1B), Jurkat (FIG. 1C),and RF33.70 (FIG. 1D) as detected by DMF10.62.3.

FIGS. 2A-D are four flow cytometric graphs showing the expression of the40 kDa protein on Day 14 fetal thymus (FIG. 2A), total adult spleen(FIG. 2B), total adult thymus (FIG. 2C) and total adult bone marrow(FIG. 2D) as detected by DMF10.62.3.

FIGS. 3A-G are seven flow cytometric graphs showing the expression ofthe 40 kDa protein on unstimulated, freshly harvested T and B cells(FIG. 3A), activated T cells at 24 hours (FIG. 3B), activated T cells at48 hours (FIG. 3C), activated T cells at 72 hours (FIG. 3D), activatedsplenic B cells at 24 hours (FIG. 3E), activated splenic B cells at 48hours (FIG. 3F) and activated splenic B cells at 72 hours (FIG. 3G) asdetected by DMF10.62.3.

FIGS. 4A-C are three line graphs showing the inhibition of spontaneousproliferation of E710.2.3 cells (FIG. 4A), RMA-S cells (FIG. 4B) orRF33.70 cells (FIG. 4C), by the monoclonal antibody DMF10.62.3.

FIGS. 5A-I are nine flow cytometric graphs showing the induction ofapoptosis in E710.2.3 cells treated with 1 μg/ml of DMF10.62.3 for 1hour (FIG. 5A), 1 μg/ml of DMF10.62.3 for 2 hours (FIG. 5B), 1 μg/ml ofDMF10.62.3 for 3 hours (FIG. 5C), Hamster IgG for 3 hours (FIG. 5D), 15μg/ml DMF10.62.3 for 1 hour (FIG. 5E), 15 μg/ml DMF10.62.3 for 2 hours(FIG. 5F), 15 μg/ml DMF10.62.3 for 3 hours (FIG. 5G), Hamster IgG for 3hours (FIG. 5H), and no antibodies (FIG. 5I).

DETAILED DESCRIPTION

The present invention features antibodies, e.g., monoclonal antibodies,that specifically bind to a 40 kDa protein. The 40 kDa protein is anovel cell surface protein expressed on a variety of types of tumorcells including thymic lymphoma, T-cell tumor, a B-cell lymphoma,melanoma, osteosarcoma, and acute T-cell leukemia and in a variety ofdifferent species including humans, monkeys, and mice. The antibodiesshow no reactivity with normal adult hemopoietic cells. Upon binding ofan antibody of the invention to a cell that expresses the 40 kDaprotein, the cell stops proliferating and undergoes apoptosis. The 40kDa protein is a novel death inducing protein based on the observationthat that when monoclonal antibodies bind to this protein on a cell, thecells undergo apoptosis.

Three hybridoma cell lines that produce monoclonal antibodies thatspecifically bind to the 40 kDa protein have been deposited with theATCC under Accession No. PTA-377 (DMF10.62.3), Accession No. PTA-405(DMF10.167.4), or Accession No. PTA-404 (DMF10.34.36).

The antibodies described herein have a variety of uses. The antibodiescan be used in in vitro diagnostic assays to determine the presence ofmalignant cells in mammalian, e.g., human, tissues. The antibodies canalso be used to localize tumors in vivo by administering to a subject anisolated antibody described herein which is labeled with a reportergroup. The antibodies also have therapeutic applications. In addition,the antibodies can be used to treat tumors or deliver an antitumoragent.

Methods of Making Antibodies

Antibodies are immunoglobulin molecules and immunologically activeportions of immunoglobulin molecules. Examples of fragments ofimmunoglobulin molecules include fragments of an antibody, e.g., F(ab)and F(ab′)₂ portions, which can specifically bind to the 40 kDa protein.Fragments can be generated by treating the antibody with an enzyme suchas pepsin. The term monoclonal antibody or monoclonal antibodycomposition refers to a population of antibody molecules that containonly one species of an antigen binding site capable of immunoreactingwith a particular epitope of a polypeptide or protein. A monoclonalantibody composition thus typically displays a single binding affinityfor the protein to which it specifically binds

Immunization

Polyclonal and monoclonal antibodies against the 40 kDa protein can beraised by immunizing a suitable subject (e.g., a rabbit, goat, mouse orother mammal) with an immunogenic preparation which contains a suitableimmunogen. Immunogens include cells such as cells from immortalized celllines E710.2.3, RMA-S, CTLL, LB 117.4, A20, WEHI-231, PBK101A2, C2.3,B16, MC57, WOP-3027, 293T, 143Btk, Jurkat, or Cos, which have all beenshown to express the novel 40 kDa protein.

Alternatively, the immunogen can be the purified or isolated 40 kDaprotein itself For example, the monoclonal antibody produced by thehybridoma cell line deposited as ATCC No. PTA-377, PTA-405, or PTA-404can be used to isolate the protein from a cell which produces theprotein, e.g., E710.2.3, RMA-S, CTLL, LB17.4, A20, WEHI-231, PBK101A2,C2.3, B16, MC57, WOP-3027, 293T, 143Btk, Jurkat, or Cos, using affinitychromatography, immunoprecipitation or other techniques which are wellknown in the art.

The antibodies raised in the subject can then be screened to determineif the antibodies bind to fetal thymocytes while not binding to adultthymocytes. Such antibodies can be further screened in the assaysdescribed herein. For example, these antibodies can be assayed todetermine if they inhibit cell proliferation of cells to which theybind; induce homotypic aggregation of cells; and/or induce apoptosis incells to which they bind. Suitable methods to identify an antibody withthe desired characteristics are described herein. For example, theability of an antibody to induce cell death upon binding to a cell canbe assayed using commercially available kits from R&D (Minneapolis,Minn.) or Pharmingen (San Diego, Calif.).

The unit dose of immunogen (e.g., the purified protein, tumor cellexpressing the protein, or recombinantly expressed 40 kDa protein) andthe immunization regimen will depend upon the subject to be immunized,its immune status, and the body weight of the subject. To enhance animmune response in the subject, an immunogen can be administered with anadjuvant, such as Freund's complete or incomplete adjuvant.

Immunization of a subject with an immunogen as described above induces apolyclonal antibody response. The antibody titer in the immunizedsubject can be monitored over time by standard techniques such as anELISA using an immobilized antigen, e.g., the 40 kDa protein describedherein.

Other methods of raising antibodies against the 40 kDa protein includeusing transgenic mice which express human immunoglobin genes (see, e.g.,Wood et al. PCT publication WO 91/00906, Kucherlapati et al. PCTpublication WO 91/10741; or Lonberg et al. PCT publication WO 92/03918).Alternatively, human monoclonal antibodies can be produced byintroducing an antigen into immune deficient mice that have beenengrafted with human antibody-producing cells or tissues (e.g., humanbone marrow cells, peripheral blood lymphocytes (PBL), human fetal lymphnode tissue, or hematopoietic stem cells). Such methods include raisingantibodies in SCID-hu mice (see Duchosal et al. PCT publication WO93/05796; U.S. Pat. No. 5,411,749; or McCune et al. (1988) Science241:1632-1639)) or Rag-1/Rag-2 deficient mice. Human antibody-immunedeficient mice are also commercially available. For example, Rag-2deficient mice are available from Taconic Farms (Germantown, N.Y.).

Hybridomas

Monoclonal antibodies can be generated by immunizing a subject with animmunogen. At the appropriate time after immunization, e.g., when theantibody titers are at a sufficiently high level, antibody producingcells can be harvested from an immunized animal and used to preparemonoclonal antibodies using standard techniques. For example, theantibody producing cells can be fused by standard somatic cell fusionprocedures with immortalizing cells such as myeloma cells to yieldhybridoma cells. Such techniques are well known in the art, and include,for example, the hybridoma technique as originally developed by Kohlerand Milstein, (1975) Nature, 256: 495497), the human B cell hybridomatechnique (Kozbar et al., (1983) Immunology Today, 4: 72), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole etal., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.pp. 77-96). The technology for producing monoclonal antibody hybridomasis well known.

Monoclonal antibodies can also be made by havesting antibody producingcells, e.g., splenocytes, from transgenic mice expressing humanimmunogloulin genes and which have been immunized with the 40 kDaprotein. The splenocytes can be immortalized through fusion with humanmyelomas or through transformation with Epstein-Barr virus (EBV). Thesehybridomas can be made using human B cell- or EBV-hybridoma techniquesdescribed in the art (see, e.g., Boyle et al., European PatentPublication No. 0 614 984).

Hybridoma cells producing a monoclonal antibody which specifically bindsto the 40 kDa protein are detected by screening the hybridoma culturesupernatants by, for example, screening to select antibodies thatspecifically bind to the immobilized 40 kDa protein, or by testing theantibodies as described herein to determine if the antibodies have thedesired characteristics, e.g., the ability to inhibit cellproliferation.

Hybridoma cells that produce monoclonal antibodies that test positive inthe screening assays described herein can be cultured in a nutrientmedium under conditions and for a time sufficient to allow the hybridomacells to secrete the monoclonal antibodies into the culture medium, tothereby produce whole antibodies. Tissue culture techniques and culturemedia suitable for hybridoma cells are generally described in the art(see, e.g., R. H. Kenneth, in Monoclonal Antibodies: A New Dimension InBiological Analyses, Plenum Publishing Corp., New York, N.Y. (1980).Conditioned hybridoma culture supernatant containing the antibody canthen be collected.

Recombinant Combinatorial Antibody Libraries

Monoclonal antibodies can be engineered by constructing a recombinantcombinatorial immunoglobulin library and screening the library with the40 kDa protein. Kits for generating and screening phage displaylibraries are commercially available (e.g., the Pharmacia RecombinantPhage Antibody System, Catalog No. 27-9400-01; and the StratageneSurfZAP Phage Display Kit, Catalog No. 240612). Briefly, the antibodylibrary is screened to identify and isolate phages that express anantibody that specifically binds to the 40 kDa protein. In a preferredembodiment, the primary screening of the library involves screening withan immobilized 40 kDa protein.

Following screening, the display phage is isolated and the nucleic acidencoding the selected antibody can be recovered from the display phage(e.g., from the phage genome) and subcloned into other expressionvectors by well known recombinant DNA techniques. The nucleic acid canbe further manipulated (e.g., linked to nucleic acid encoding additionalimmunoglobulin domains, such as additional constant regions) and/orexpressed in a host cell.

Chimeric and Humanized Antibodies

Recombinant forms of antibodies, such as chimeric and humanizedantibodies, can also be prepared to minimize the response by a humanpatient to the antibody. When antibodies produced in non-human subjectsor derived from expression of non-human antibody genes are usedtherapeutically in humans, they are recognized to varying degrees asforeign, and an immune response may be generated in the patient. Oneapproach to minimize or eliminate this immune reaction is to producechimeric antibody derivatives, i.e., antibody molecules that combine anon-human animal variable region and a human constant region. Suchantibodies retain the epitope binding specificity of the originalmonoclonal antibody, but may be less immunogenic when administered tohumans, and therefore more likely to be tolerated by the patient.

Chimeric monoclonal antibodies can be produced by recombinant DNAtechniques known in the art. For example, a gene encoding the constantregion of a non-human antibody molecule is substituted with a geneencoding a human constant region (see Robinson et al., PCT PatentPublication PCT/US86/02269; Akira, et al., European Patent Application184,187; or Taniguchi, M., European Patent Application 171,496).

A chimeric antibody can be further “humanized” by replacing portions ofthe variable region not involved in antigen binding with equivalentportions from human variable regions. General reviews of “humanized”chimeric antibodies are provided by Morrison, S. L. (1985) Science,229:1202-1207 and by Oi et al: (1986) BioTechniques, 4-214. Such methodsinclude isolating, manipulating, and expressing the nucleic acidsequences that encode all or part of an immunoglobulin variable regionfrom at least one of a heavy or light chain. The cDNA encoding thehumanized chimeric antibody, or fragment thereof, can then be clonedinto an appropriate expression vector. Suitable “humanized” antibodiescan be alternatively produced by (complementarity determining region(CDR) substitution (see U.S. Pat. No. 5,225,539; Jones et al. (1986)Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1.534; andBeidler et al. (1988) J. Immunol. 141:4053-4060).

Epitope imprinting can also be used to produce a “human” antibodypolypeptide dimer that retains the binding specificity of the hamsterantibodies specific for the 40 kDa protein produced by the hybridomadeposited as ATCC No. PTA-377, ATCC No. PTA-405, or ATCC No. PTA-404.Briefly, a gene encoding a non-human variable region (VH) with specificbinding to an antigen and a human constant region (CH1), is expressed inE. coli and infected with a phage library of human VλCλ genes. Phagedisplaying antibody fragments are then screened for binding to the 40kDa protein. Selected human VX genes are recloned for expression of VλCλchains and E. coli harboring these chains are infected with a phagelibrary of human VHCH1 genes and the library is subject to rounds ofscreening with antigen coated tubes. See Hoogenboom et al. PCTpublication WO 93/06213.

Antibody Fragments

The present invention encompasses new antitumor antibodies and anyfragments thereof containing the active binding region of the antibody,such as Fab, F(ab′)2, and Fv fragments. Such fragments can be producedfrom the antibody using techniques well established in the art (see,e.g., Rousseaux et al., in Methods Enzymol., 121:663-69 Academic Press,(1986)). For example, the F(ab′)₂ fragments can be produced by pepsindigestion of the antibody molecule, and the Fab fragments can begenerated by reducing the disulphide bridges of the F(ab′)₂ fragments.

Utility of Antibodies

The antibodies described herein have a variety of uses. The antibodiescan be used in vitro for diagnostic purposes to determine the presenceof malignant cells in human tissues. The method involves examining atissue sample for the presence of the 40 kDa protein. For example, thetissue sample can be contacted with the monoclonal antibody produced bythe hybridoma cell line ATCC NO: PTA-377, ATCC No. PTA-405, or ATCC No.PTA-404, and the ability of the antibody to specifically bind to thecells in the tissue sample is determined. Binding indicates the presenceof a tumor cell. Alternatively, the antibody can also be used to screenblood samples for released antigen.

The antibodies can also be used to localize a tumor in vivo byadministering to a subject an isolated antibody of the present inventionwhich is labeled with a reporter group which gives a detectable signal.The bound antibodies are then detected using external scintigraphy,emission tomography, or radionuclear scanning. The method can be used tostage a cancer in a patient with respect to the extent of the diseaseand to monitor changes in response to therapy.

The antibodies also have therapeutic applications. The new antibodiescan be used to treat tumors, because specific binding of the antibody tothe tumor cell causes the cell to stop proliferating and to die.

The antibodies can also be used therapeutically, e.g., as targetingagents, to deliver antitumor agents to the tumors. Such anti-tumoragents include chemotherapeutic drugs, toxins, immunological responsemodulators, enzymes, and radioisotopes.

Detectable Labels

The antibodies that react with the 40 kDa protein can be useddiagnostically, e.g., to detect the presence of a tumor in a subject.Detection can be facilitated by coupling the antibody to a detectablelabel. Examples of detectable labels include various enzymes, prostheticgroups, fluorescent materials, luminescent materials, bioluminescentmaterials, electron dense labels, labels for MRI, and radioactivematerials. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examplesof suitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoeryhrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

Antibodies as Targeting Agents

The antibodies and antibody fragments described herein can be conjugatedto a moiety and the antibody can be used to direct the moiety to thesite of a tumor cell which expresses the 40 kDa protein. Examples ofmoieties include, toxins, radionuclides, or chemotherapeutic agentswhich can be used to kill tumor cells, or imaging agents which can beused to locate and size tumors expressing the 40 kDa protein. Theantibodies used to direct the moiety to the tumor in humans arepreferably monoclonal antibodies, e.g., a humanized monoclonalantibodies.

The antibody can be fused to the moiety, e.g., the toxin, either byvirtue of the moiety and the antibody being encoded by a fused genewhich encodes a hybrid protein molecule, or by means of conjugation,e.g., a non-peptide covalent bond, e.g., a non-amide bond, which is usedto join separately produced antibody and the moiety.

The antibody described herein can also be fused to another antibody thatis specific for immune cells and stimulates the immune cells to kill thetumor.

Toxins

Useful toxin molecules include peptide toxins, which are significantlycytotoxic when present intracellularly. Examples of toxins includecytotoxins, metabolic disrupters (inhibitors and activators) thatdisrupt enzymatic activity and thereby kill tumor cells, and radioactivemolecules that kill all cells within a defined radius of the effectorportion. A metabolic disrupter is a molecule, e.g., an enzyme or acytokine, that changes the metabolism of a cell such that its normalfunction is altered. Broadly, the term toxin includes any effector thatcauses death to a tumor cell.

Many peptide toxins have a generalized eukaryotic receptor bindingdomain; in these instances the toxin must be modified to prevent killingcells not bearing the targeted protein (e.g., to prevent killing ofcells not bearing the 40 kDa protein but having a receptor for theunmodified toxin). Such modifications must be made in a manner thatpreserves the cytotoxic function of the molecule. Potentially usefultoxins include, but are not limited to: diphtheria toxin, cholera toxin,ricin, O-Shiga-like toxin (SLT-I, SLT-II, SLT-II_(v)), LT toxin, C3toxin, Shiga toxin, pertussis toxin, tetanus toxin, Pseudomonasexotoxin, alorin, saponin, modeccin, and gelanin. Other toxins includetumor necrosis factor alpha (TNF-a) and lymphotoxin (LT). Another toxinwhich has antitumor activity is calicheamicin gamma 1, a diyne-enecontaining antitumor antibiotic with considerable potency against tumors(Zein, N., et al., Science, 240:1198-201 (1988)).

As an example, diphtheria toxin can be conjugated to the antibodiesdescribed herein. Diphtheria toxin, whose sequence is known, isdescribed in detail in Murphy, U.S. Pat. No. 4,675,382, which isincorporated herein by reference. The natural diphtheria toxin moleculesecreted by Corynebacterium diphtheriae consists of several functionaldomains that can be characterized, starting at the amino terminal end ofthe molecule, as enzymatically-active Fragment A (amino acidsGly₁-Arg₁₉₃) and Fragment B (amino acids Ser₁₉₄-Ser₅₃₅), which includesa translocation domain and a generalized cell binding domain (amino acidresidues 475 through 535).

Linkage of Toxins to Antibodies

The antibody and the toxin moiety can be linked in any of several ways.If the compound is produced by expression of a fused gene, a peptidebond serves as the link between the cytotoxin and the antibody.Alternatively, the toxin and the antibody can be produced separately andlater coupled by means of a non-peptide covalent bond. For example, thecovalent linkage may take the form of a disulfide bond. In this case,the DNA encoding this antibody can be engineered, by conventionalmethods, to contain an extra cysteine codon.

For a disulfide bond linkage, the toxin molecule is also derivatizedwith a sulfhydryl is group reactive with the cysteine of the modifiedantibody. In the case of a peptide toxin this linkage can beaccomplished by inserting a cysteine codon into the DNA sequenceencoding the toxin. Alternatively, a sulfhydryl group, either by itselfor as part of a cysteine residue, can be introduced using solid phasepolypeptide techniques. For example, the introduction of sulfhydrylgroups into peptides is described by Hiskey, Peptides, 3:137 (1981).

Derivatization can also be carried out according to the method describedfor the derivatization of a peptide hormone in Bacha et al., U.S. Pat.No. 4,468,382. The introduction of sulfhydryl groups into proteins isdescribed in Maasen et al., Eur. J. Biochem., 134:32 (1983).

Once the required sulfhydryl groups are present, the cytotoxin and theantibody are purified, both sulfur groups are reduced, cytotoxin andantibody are mixed (in a ratio of about 1:5 to 1:20), and disulfide bondformation is allowed to proceed to completion (generally 20 to 30minutes) at room temperature. The mixture is then dialyzed againstphosphate buffered saline to remove unreacted antibody and toxinmolecules. Sephadex^(R) chromatography or the like is used to separatethe desired toxin-antibody conjugate compounds from toxin-toxin andantibody-antibody conjugates on the basis of size

Immune Response Modulators

The antitumor moiety can also be a modulator of the immune system thateither activates or inhibits the body's immune system at the locallevel. For example, cytokines, e.g., lymphokines such as IL-2, deliveredto a tumor can cause the proliferation of cytotoxic T-lymphocytes ornatural killer cells in the vicinity of the tumor

Radioactive Molecules

The moiety or reporter group can also be a radioactive molecule, e.g., aradionucleotide, or a so-called sensitizer, e.g., a precursor molecule,that becomes radioactive under specific conditions, e.g., boron whenexposed to a beam of low-energy neutrons, in the so-called “boronneutron capture therapy” (BNCT). Barth et al., Scientific American,October 1990:100-107 (1990). Compounds with such radioactive effectorportions can be used both to inhibit tumor cell proliferation and tolabel the tumor cells for imaging purposes.

Radionuclides are single atom radioactive molecules that can emit eitherα, β, or γ particles. Alpha particle emitters are preferred to β or γparticle emitters, because they release far higher energy emissions overa shorter distance, and are therefore efficient without significantlypenetrating, and harming, normal tissues. Suitable a particle emittingradionuclides include ²¹¹At, ²¹²Pb, and ²¹²Bi.

The radioactive molecule must be tightly linked to the antibody eitherdirectly or by a bifunctional chelate. This chelate must not allowelution and thus premature release of the radioactive molecule in vivo.Waldmann, Science, 252:1657-62 (1991).

To adapt BNCT to the present invention, a stable isotope of boron, e.g.,boron 10, is selected as the antitumor moiety or effector portion of thecompound. The boron is delivered to and concentrates in or on the tumorcells by the specific binding of the antibody to the tumor cell. After atime that allows a sufficient amount of the boron to accumulate, thetumor is imaged and irradiated with a beam of low-energy neutrons,having an energy of about 0.025 eV. While this neutron irradiation, byitself, causes little damage to either the healthy tissue surroundingthe tumor, or the tumor itself, boron 10 (e.g., on the surface of atumor cell) captures the neutrons, thereby forming an unstable isotope,boron 11. Boron 11 instantly fissions yielding lithium 7 nuclei andenergetic a particles, about 2.79 million Ev. These heavy particles area highly lethal, but very localized, form of radiation, becauseparticles have a path length of only about one cell diameter (10microns).

Calculations have shown that to destroy a tumor cell, about one billionboron atoms are required along with a flow of thermal neutrons of from10¹² to 10¹³ neutrons per square centimeter, so that the radiationgenerated by the a particles exceeds the background radiation generatedby neutron capture reactions with nitrogen and hydrogen.

Imaging Moieties

The antibodies described herein specifically bind to the 40 kDa proteinand are thus also useful to detect human tumors. One such approachinvolves the detection of tumors in vivo by tumor imaging techniquesusing the antibody labeled with an appropriate moiety or reporter group,e.g., an imaging reagent that produces a detectable signal. Imagingreagents and procedures for labeling antibodies with such reagents arewell known (see, e.g., Wensel and Meares, Radio Immunoimaging andRadioimmunotherapy, Elsevier, New York (1983); Colcher et al., Meth.Enzymol., 121:802-16 (1986)). The labeled antibody can be detected by atechnique such as radionuclear scanning (see, e.g., Bradwell et al. inMonoclonal Antibodies for Cancer Detection and Therapy, Baldwin et al.(eds.), pp. 65-85, Academic Press (1985)).

Administration

The antibodies described herein can be administered to a subject, e.g.,an animal or a human, to image or treat tumors. The antibodies can beadministered alone, or in a mixture, e.g., in the presence of apharmaceutically acceptable excipient or carrier (e.g., physiologicalsaline). The excipient or carrier is selected on the basis of the modeand route of administration. Suitable pharmaceutical carriers aredescribed in Remington's Pharmaceutical Sciences (E. W. Martin), a wellknown reference text in this field, and in the USP/NF (United StatesPharmacopeia and the National Formularly).

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. Examples of routes of administrationinclude parenteral, e.g., intravenous, intradermal, subcutaneous, oral(e.g., inhalation), transdermal (topical), transmucosal, and rectaladministration. Solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes, or multiple dose vials made of glass or plastic.

The most effective mode of administration and dosage regimen for thecompositions of this invention depend upon the severity and course ofthe disease, the patient's health and response to treatment, and thejudgment of the treating physician. Accordingly, the dosages of thecompositions should be titrated to the individual patient. An effectivedose of the antibody compositions of this invention is in the range offrom about 1 ug to about 5000 mg, preferably about 1 to about 500 mg, orpreferably about 100-200 mg.

Diagnostic Kits

The invention also encompasses diagnostic kits for carrying out themethods disclosed above. The diagnostic kit includes (a) a monoclonalantibody described herein, and (b) a conjugate of a specific bindingpartner for the antibody and a label for detecting bound antibody. Thekit may also include ancillary agents such as buffering agents andprotein stabilizing agents, e.g., polysaccharides and the like. Thediagnostic kit may further include, where necessary, other components ofa signal-producing system including agents for reducing backgroundinterference, control reagents, and an apparatus for conducting a test.In another embodiment, the diagnostic kit includes a conjugate of amonoclonal antibody of the invention and a label capable of producing adetectable signal. Ancillary agents as mentioned above may also bepresent. Instructions on how to use the diagnostic kit are generallyalso included.

Polypeptides

The monoclonal antibodies described herein can be used to isolate andcharacterize the 40 kDa protein to which they bind. The proteinrecognized by the monoclonal antibody is a novel cell surface proteinfound on a variety of tumor cells including thymic lymphoma, T-celltumor, a B-cell lymphoma, melanoma, osteosarcoma, and acute T-cellleukemia. The protein is a death inducing molecule based on theobservation that when monoclonal antibodies bind to this protein, thecell on which the protein is expressed dies.

The protein recognized by the monoclonal antibodies of the invention canbe isolated from cells expressing the protein (e.g., E710.2.3, RMA-S,CTLL, LB17.4, A20, WEHI-23 1, PBK101A2, C2.3, B16, MC57, WOP-3027, 293T,143Btk, Jurkat, or Cos). For example, the monoclonal antibodiesdescribed herein can be used to immunoprecipitate the protein. Todetermine the sequence of the protein, the protein can be purified bySDS-PAGE, electroblotted onto an Immobilon membrane (Millipore Corp.,Bedford, Mass.), and the membrane stained with Coomassie Brilliant Blue.The stained protein band (Mr=40 kDa) can then be excised with a razorblade for subsequent amino-terminal sequence analysis. Amino-terminalsequence analysis, such as automated Edman degradation, are well knownin the art.

The invention also features fusion proteins that include the 40 kDaprotein fused to an unrelated protein. The unrelated protein can beselected to facilitate purification, detection, solubilization, or toprovide some other function. Fusion proteins can be producedsynthetically, or the protein can be linked to an unrelated proteinusing an appropriate coupling reagent, e.g., dicyclohexylcarbodiimide(DCC). Alternatively, fusion proteins can be produced recombinately bycloning a nucleotide sequence which expresses the fusion protein into anappropriate expression vector. The recombinant fusion polypeptide canthen be purified from the culture medium or from lysates of the cells.

The 40 kDa protein is useful, e.g., as a vaccine to immunize againstcertain tumors. Procedures for preparing such vaccines are known in theart (see, e.g., Estin et al., Proc. Nat'l. Acad. Sci. (USA), 85:1052(1988)). Briefly, recombinant viruses are constructed for expression ofthe cloned tumor-associated protein. Cells infected with the recombinantviruses will express the protein at the surface of the cells togetherwith the host's histocompatibility antigens and immunogenic viralproteins. This favors the induction of cellular immunity which plays akey role in tumor rejection.

The invention also provides a method for identifying modulators, i.e.,test compounds or agents (e.g., peptides, peptidomimetics, smallmolecules or drugs) which bind to the 40 kDa protein or which have astimulatory or inhibitory effect on expression or activity of the 40 kDaprotein. For example, an antagonist of the 40 kDa protein would beuseful for inhibiting apoptosis in a cell. This antagonist might play arole in inhibiting abherant apoptosis in a subject.

EXAMPLES Example 1 Generation of Antitumor Antibodies

In an attempt to identify novel functional molecules that may beinvolved in the growth or survival of lymphomas and/or in normalthymocyte function, hybridomas from hamsters injected with E710.2.3 weregenerated as follows. Armenian hamsters were injected intraperitoneallywith 10 million E710.2.3 and boosted 7-10 times before fusion. Fusionswere performed using the fusion partner P3X63-AG8.653 as described(Schreiber et al. (1985) Immunol 134:1609). Supernatants from hybridswere first screened using immunofluorescence and flow cytometry for theability to bind to E710.2.3A. One particular antibody, referred toherein as DMF10.62.3, stained the surface of E710.2.3 brightly.

Immunofluorescence analysis revealed that DMF10.62.3 reacted with anumber of murine cell lines (Table I), but was absent from others (TableII). Positive cell lines included some T cell lines (e.g. RMA-S),several B cell lymphomas (e.g. A20 and WEHI-231), and a macrophage cellline (C2.3). DMF10.62.3 also specifically bound to several immortalizedcells of non-hematopoietic origin, inducing a stromal cell line(PBK101A2), a melanoma (B16), a sarcoma (MC57), and apolyoma-transformed fibroblast (WOP-3027). Several other immature (e.g.,G58.2) and mature T cells (e.g., EL4), a macrophage (e.g., A3.1), adendritic cell (DC2.4), and a fibroblast cell line (LADp31) werenegative for DMF10.62.3. Interestingly, the mAb also reacted withseveral human immortalized cell lines, including Jurkat, 293T and143Btk- and also with a monkey SV40-transformed kidney cell line, Cos7.DMF10.62.3 did not bind to certain human cell lines, such as the Blymphoblastoid cell, 721, and the cervical carcinoma cell HeLa. Stainingpatterns of representative cell lines are shown in FIG. 1 for theDMF10.62.3-positive cells E710.2.3 (FIG. 1A), A20 (FIG. 1B) and Jurkat(FIG. 1C) and the DMF10.62.3-negative cell, RF33.70 (FIG. 1D).

TABLE I CELL LINE DESCRIPTION E710.2.3 Murine thymic lymphoma RMA-SMurine T cell tumor CTLL Murine IL-2 dependent T cell line LB27.4 MurineB cell hybridoma A20 Murine B cell lymphoma WEHI-231 Murine B celllymphoma PBK101A2 Murine thymic stromal cell line C2.3 Murineimmortalized bone marrow macrophage B16 Murine melanoma MC57 Murinemethylcholanthrene-induced tumor WOP-3027 Murine polyoma-transformedfibroblast 293T Human transformed primary embryonal kidney 143Btk- Humanosteosarcoma

The above data indicated that new antibodies specifically bind to manybut not all immortalized cell lines, and that binding is not species orcell lineage-restricted.

TABLE II CELL LINE DESCRIPTION RF33.70 Murine T-T hybrid DO11.10 MurineT-T hybrid 13G7.3.2 Murine T-T hybrid HT-2 Murine IL-2 dependent T cellline EL-4 Murine T cell lymphoma G58.2 Murine thymic lymphoma NFC105Murine thymic lymphoma P815 Murine mastocytoma P388D1 Murinemonocyte/macrophage tumor LAD_(p)31 Mouse L cell line A3.1 Murineimmortalized bone marrow-derived macrophage DC2.4 Murine immortalizeddendritic cell line 721 Human B cell line HeLa Human epithelial cervicalcarcinoma E36 Hamster lung carcinoma BHK-21 Hamster kidney cell line CHOChinese hamster ovary

Example 2 Expression of the Molecule Recognized by DMF10.62.3

Expression of the molecule recognized by DMF10.62.3 was examined infetal thymocytes as follows. Timed pregnancies of C57BI/10 mice producedembryos that were sacrificed at fetal day 14. The fetal thymi wereharvested in PBS using an Eppendorf tube glass plunger. Single cellsuspensions were incubated for 20 minutes on ice with an anti-Fc gammareceptor II/III (Pharmingen) to block Fc receptors. Cells weresubsequently stained with either DMF10.62.3 or hamster IgG for 30minutes followed by FITC conjugated goat anti-hamster, along withallophycoyanin (APC)-conjugated anti-Thy 1.2 (Pharmingen). In someexperiments, anti-CD25 conjugated to PE and anti-CD44 conjugated toCy-Chrome (Pharmingen) were also included. The stained cells were fixedovernight in 1% paraformaldehyde and subsequently analyzed by flowcytometry.

Results showed that day 14 fetal thymocytes stained with DMF10.62.3 werepositive for the 40 kDa protein and the protein was found to be presenton Thy 1.2 positive cells (FIG. 2A). Interestingly, the protein waspresent on both CD25⁺CD44⁺ fetal thymocytes as well as CD44⁺CD25⁻ fetalthymocytes. However, staining of adult thymus (FIG. 2C), adult spleen(FIG. 2B). and adult bone marrow cells (FIG. 2D) showed that the proteinrecognized by DMF10.62.3 is not present on any of these cells at levelsabove those seen with control hamster IgG. Furthermore, the proteincould not be detected on adult CD4⁻CD8⁻ thymocytes after gating onCD4⁻CD8⁻ cells in a multiparameter analysis, by flow cytometry oranalysis of this population from RAG−/−mice. Day 14 fetal liver cellswere also non-reactive with DMF10.62.3.

To determine if the protein recognized by DMF10.62.3 was present onnormal, activated cells, splenic T cells were activated with the T cellmitogen ConA and stained for expression of the protein recognized byDMF10.62.3 as follows. Spleen, thymus, and bone marrow cells wereprepared from adult (4-6 months old) Balb/c or C57B1/6 mice. Red bloodcorpuscles were removed from spleen cells suspensions using trisammonium chloride lysis. Unstimulated cells were stained immediately.Lymphoblasts were stimulated in culture with 1 μg/ml of ConA or 10 μg/mlof LPS. After 1-3 days of culture, cells were stained for the expressionof DMF10.62.3. No significant staining above background was seen inunstimulated cells (FIG. 3A) or cells stimulated with ConA at 24 (FIG.3B), 48. (FIG. 3C), or 72 (FIG. 3D) hours after activation (no shift inFACs profile). As a positive control, ConA stimulated cells were stainedwith CD25. As expected, ConA treatment resulted in a significantincrease in expression of CD25 on these cells as compared tounstimulated cells (binding of CD25 to cells causes shift of FAC'sprofile). Similarly, when splenic B cells were activated withlipopolysaccaride (LPS), no staining with DMF10.62.3 was seen at 24(FIG. 3E), 48 (FIG. 3F), and 72 (FIG. 3G) hours (no shift in FAC'sprofile), whereas these cells did express CD25 (shift in FAC's profile).The protein recognized by DMF10.62.3 is not present on adult bone marrowcells (FIG. 2D). These data indicate that the protein recognized byDMF10.62.3 is present on some fetal thymocytes, but not on normalquiescent or activated cells of hematopoietic origin in adult animals.

Example 3 DMF10.62.3 Inhibits Proliferation of Tumor Cells

When grown at low density, and maintained in the absence of PMA,E170.2.3 cells proliferate slowly or not at all. However theyproliferate when cocultured with thymocytes. DMF10.62.3 was initiallyidentified by its ability to block this thymocyte-induced proliferation.As shown in Table III, DMF10.62.3 completely inhibits this response(FIG. 4A). Proliferation assays were conducted as follows. E10.2.3 cellswere washed free of PMA and cultured in complete RPMI for 48 hours atlow cell density (<10⁵/ml), to reduce background proliferation.Subsequently, 5×10³ cells were cultured for 72 hours in flat bottommicrotiter plates with 25 ng/ml PMA or 5×10⁵ thymocytes in the presenceor absence of antibodies. In experiments examining the effects ofantibodies on the spontaneous proliferation of cells, E710.2.3 (grown athigh density>10⁵/ml) or RMA-S cells were cultured for 36 hours in thepresence or absence of different concentrations of antibody.³H-thymidine (1 TCi/well) was added for the last 5 hours and theincorporation of label into DNA was measured in a J-scintillationcounter (Wallac, Gaithersburg, Md.).

TABLE III Medium Thymocytes PMA Control 8,699 33,802 5 4,271 (noantibody) DMF10.62.3 938 750 450 DMF10.132 6,646 27,156 25,216

The ability of DMF10.62.3 to inhibit the response of E710.2.3 to otherstimuli was also investigated. As shown in Table III, the antibody alsoblocked PMA-induced proliferation of E710.2.3. Moreover, E710.2.3spontaneously proliferated when grown at high density, and DMF10.62.3inhibited this response (FIG. 4A). Proliferation is significantlyinhibited at 3 μg/ml and complete inhibition is observed at 12.5 μg/ml.In contrast, hamster IgG had no effect on the response of E710.2.3 (FIG.4A) to any of these stimuli. Similarly, many of the mAbs from theoriginal fusion bound to E710.2.3, but did not inhibit its proliferation(e.g. DMF10.132) (Table III). Therefore, DMF10.62.3 specificallyinhibited the proliferation of E710.2.3 regardless of the stimulus usedto induce proliferation.

The protein recognized by DMF10.623 is present on a number of other celllines. Therefore it was of interest to determine if the antibody had asimilar effect on their spontaneous proliferation. DMF10.62.3 inhibitedthe proliferation of RMA-S (FIG. 4B), as well as a number of other celllines tested. In contrast, the antibody had no effect on the spontaneousproliferation of RF33.70, which was negative for the presence of theDMF10.62.3 protein (FIG. 4C).

Example 4 DMF10.62.3 Induces Cell Death by Apoptosis

Apoptosis was assayed using kits from R&D (Minneapolis, Minn.) andPharmingen (San Diego, Calif.). Briefly, 2×10⁵ cells were incubated withvarious concentrations of antibody in 200 it medium. At the end of theincubation, cells were washed 2× in PBS, treated with PI and FITCannexin for 15 minutes, and then analyzed by flow cytometry. DNAfragmentation was assessed by agarose gel electrophoresis on 2% agarosegels as described by Schattner et al. ((1995) J. Exp. Med. 182:1557).

Cultures of cells treated with antibody DMF10.62.3 were visuallyinspected and the number of intact cells was noted to decrease. Inaddition, the cells no longer excluded the vital dye trypan blue. Thisobservation, as well as the inhibition of proliferation, suggested thatthe antibody was cytotoxic to the cells. Therefore, studies wereperformed to determine the mechanism by which DMF10.62.3 was inducingcell death.

Cells can die by apoptosis or necrosis. One of the early changes seen incells undergoing apoptosis is the externalization of phosphatidylserineon the plasma membrane, and this can be detected by staining withFITC-annexin. Early in the process, the apoptotic cells can excludevital dyes, such as propidium iodide, and therefore can be identified asFITC-annexin positive and PI-negative. Later in the apoptotic process,membrane integrity is lost and the FITC-annexin positive cells becomePI-positive. In contrast, during necrosis cells lose membrane integrityand become simultaneously PI-positive and FITC-annexin positive, withouta FITC annexin positive and PI-negative stage.

In the FACs profiles of FIG. 5A to 5I, cells in the left lower quadrantare live cells, cells in the lower right quadrant are undergoingapoptosis (FITC-annexin positive), and cells in the upper right quadrantare cells that are dead by apoptosis and/or necrosis (PI-positive andFITC-annexin positive). The percentage of cells in each quadrant is alsoshown. In the present study, a percentage of E710.2.3 cells underwentspontaneous apoptosis in culture (10.9 to 15% Annexin+, PI−). However,as little as 1 μg/ml of DMF10.62.3 caused a significant increase inapoptosis in 1 hour (28.9% Annexin+, PI−; FIG. 5A), and this apoptosisincreased over time (48.6% Annexin+, PI− positive by 3 hours) (FIG. 5C).Higher amounts of DMF10.62.3 (15 μg/ml)-stimulated apoptosis morequickly in time (37.1% Annexin+, PI− positive by 1 hour) and in morecells (FIG. 5E-G). In contrast, treatment with similar amounts ofhamster IgG had no significant effect above that of medium alone (FIGS.5D, 5H, 5I). Apoptosis was also verified by visualizing DNAfragmentation by agarose gel electrophoresis.

Since the protein recognized by DMF10.62.3 was expressed on other cells,and this antibody inhibited their proliferation (where tested), it wasfurther investigated whether DMF10.62.3 also stimulated them to undergoapoptosis. DMF10.62.3 caused significant apoptosis of the murine cellslines RMA-S, CTLL, LB27.4 and A20 and the human cell lines Jurkat and143BTK-(Table IV). Apoptosis was induced using 15 μg/ml of DMF10.62.3and increased with higher concentrations of antibody. In contrast,DMF10.62.3 did not cause apoptosis in RF33.70, which is negative for theprotein. The level of apoptosis induced by DMF10.62.3 varied amongdifferent cell lines and appears to be dependent on the level of surfaceexpression as well the percentage of cells within the populationexpressing the protein (Table IV). For example, most E710.2.3 and RMAcells, expressed the protein at high levels and DMF10.62.3 induced highlevels of apoptosis in both of these cell lines. In contrast, few A20and LB27.4 cells expressed the 40 kDa protein at lower levels andDMF10.62.3 induced lower levels of apoptosis in these cells (Table IV).The stimulation of apoptosis by DMF10.62.3 appears to be independent offas, as E710.2.3 and RMA-S cells do not express fas (Table IV).

TABLE IV % Apoptotic cells Fluorescence % cells Staining for Intensitystaining murine fas Hamster No DMF10.62.3/ positive for surface CellLine DMF10.62.3 IgG treatment Hamster IgG DMF10.62.3 expression Mouse Tcell lines E710.2.3 86.9 19.8 24.7 27.5 72.6 − RMA-S 69.6 12.2 14.0 14.560.6 − CTLL 36.4 14.8 16.7 8.88 51.5 − RF33.70 8.1 8.6 7.8 0.98 0.48 −Mouse B cell lines LB27.4 16.8 7.1 10.8 1.48 7.0 + A20 16.2 13.8 12.23.3 24.8 + Human cell lines JURKAT 33.9 12.6 11.8 16.18 49.2 ND 143BTK-29.7 21.1 20.5 3.8 29.5 ND

Example 5 DMF10.62.3 Causes Homotypic Aggregation in E710.2.3 and OtherCell Lines

Homotypic aggregation is a biologically active process whereby cells arestimulated to adhere to one another. Aggregation assays were set up asfollows.

10⁵ cells were incubated with various concentrations of DMF10.62.3 orhamster IgG or without antibody in 200 it complete RPMI. To test theeffect of inhibitors, 10⁵ cells were preincubated with inhibitor for 30minutes and then DMF10.62.3 mAb (10 μg/ml) was added in the continuedpresence of inhibitor for 6 hours. To test the effect ofparaformaldehyde on aggregation, cells were fixed in 1%paraforinaldehyde for 10 minutes, washed, and then DMF10.62.3 was addedfor 6 hours. Aggregation was scored visually. Photomicrographs weretaken at 6 hours using a thermoelectrically cooled charged-coupleddevice (CCD) camera (Princeton Instruments, Trenton, N.J.).

DMF10.62.3 was found to induce homotypic aggregation of E710.2.3 inculture. At 6 hours, significant aggregation was observed with cellstreated with 5 μg/ml or more of antibody. In contrast, no aggregationwas observed in cultures treated with hamster IgG or medium. Thisaggregation was blocked by treatment with various agents includingcytochalasin B, which disrupted actin microfilaments, trifluoperazine,which inhibits calmodulin dependent processes, Na azide+2 deoxyglucose,which inhibits ATP synthesis, and EDTA which chelates Ca2+ and Mg2+. Incontrast, aggregation was not affected by colchicine, which inhibitedmicrotubule formation. The aggregation was also inhibited by incubationat 4° C. and by treatment with paraformaldehyde (Table V). These resultsindicate that the aggregation is an active process and is not simplyagglutination.

TABLE V HOMOTYPIC CHEMICAL USED EFFECT ON CELL ADHESION Cytochalasin BCytoskeleton (disrupts actin − (20 μg/ml) microfilament integricyColchicine Inhibits microtubule + (20 μg/ml) formation TrifluoperazineInhibits calmodulin dependent − (20 μM) processes Na azide (0.1%) +Inhibits ATP synthesis − 2-deoxyglucose (5 mM) EDTA (10 mM) chelatesCa2+ and Mg2+ − Medium only CONTROL (no effect) + 4° C. −Paraformaldehyde −

DMF10.62.3 also caused homotypic aggregation of some of the other celllines (e.g. RMA-S, CTLL) which express the DMF10.62.3 binding protein.However, little aggregation above the background was seen for some otherDMF10.62.3 positive cell lines (e.g. Jurkat, LB27.4, A20, 143Btk−). Noaggregation was seen with RF33.70, to which DMF10.62.3 does not bind.

The aggregation assay can be used to help determine whether a newantibody is one of the new antitumor antibodies of the invention.

Example 6 DMF10.62.3 Immunoprecipitates a 40 kDa

Protein which is not GPI-Linked

To characterize the protein bound by DMF10.62.3, ³⁵S labeling andimmunoprecipitation were preformed as follows. 5×10⁶ E710.2.3 cells werestarved for 1 hour in methionine-free medium and then incubated for 2hours with ³⁵S methionine at 0.5 mCi/ml. Labeled cells were lysed inimmunoprecipitation buffer as described by Townsend et al. ((1990) J.Immunol. 146:2235). Clarified lysates were precleared with hamster IgG,immunoprecipitated with DMF10.62.3 bound to Protein-A-sepharose, andanalyzed by SDS-polyacrylamide gel electrophoresis on 14% gels. Todetermine the molecular weight of the DMF10.62.3 binding protein,E170.2.3 cells were labeled for 2 hours with ³⁵S methionine.Immunoprecipitates from labeled cells were analyzed by SDS-PAGE underreducing conditions.

The mAb DMF10.62.3 immunoprecipitated an approximately 40 kDa proteinfrom E710.2.3 under reducing conditions. The electrophoretic mobility ofthis protein was not altered under non-reducing conditions. This bandwas not seen in immunoprecipitates with normal hamster IgG, or inimmunoprecipitates with an anti-MHC class I antibody, Y-3. A 40kDa-protein was also identified in lysates of surface labeled E710.2.3and RMA-S cells.

Several cell surface molecules such as Thy-1 and Ly-6 A/E are linked tothe cell surface via glycosylphoshatidylinositol (GPI) anchors. Thissurface linkage is sensitive to treatment with PI-PLC. To determine ifthe protein recognized by DMF10.62.3 was GPI-linked, RMA-S cells whichexpress the protein on the cell surface, were treated with PI-PLC.PI-PLC treatment did not reduce DMF10.62.3 staining but did decreasestaining for the, GPI-linked molecule Thy-1; suggesting that the 40 kDaprotein recognized by DMF10.62.3 is not anchored to the cell surface bya GPI.

Example 7 The In Vivo Effect of the DMF62.3 Antibody

AKR mice were injected IV or IP with 5×10⁶ syngeneic E710.2.3 tumorcells and received saline or an injection IP of 0.5 mg of control orDMF62.3 antibody on the initial day and again 10 days later. Thesurvival of animals was followed for 50 days (Table VI).

TABLE VI Treatment Survival Average time to death 1. saline 0% 35 days2. control antibody 0% 33 days 3. DMF62.3 100%  (No deaths)

Deposit Statement

The hybridoma cell line producing the monoclonal antibody DMF10.62.3,was received by the American Type Culture Collection (ATCC), 10801University Boulevard, Manassas, Va., on Jul. 20, 1999, and the hybridomacell lines producing the monoclonal antibodies DMF10.167.4 andDMF10.34.36 were received by the American Type Culture Collection(ATCC), 10801 University Boulevard, Manassas, Va., on Jul. 22, 1999. Thehybridomas have been deposited under conditions that assure that accessto the hybridomas will be available during the pendency of the patentapplication disclosing them to one determined by the Commissioner ofPatents and Trademarks to be entitled thereto under 37 CFR 1.14 and 35USC 122. The deposits are available as required by foreign patent lawsin countries wherein counterparts of the subject application, or itsprogeny, are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

Further, the subject culture deposits will be stored and made availableto the public in accord with the provisions of the Budapest Treaty forthe Deposit of Microorganism, i.e., they will be stored with all thecare necessary to keep them viable and uncontaminated for a period of atleast five years after the most recent request for the furnishing of asample of the deposits, and in any case, for a period of at least 30(thirty) years after the date of deposit or for the enforceable life ofany patent which may issue disclosing the cultures plus five years afterthe last request for a sample from the deposit. The depositoracknowledges the duty to replace the deposit should the depository beunable to furnish a sample when requested, due to the condition of thedeposits. All restrictions on the availability to the public of thesubject culture deposit will be irrevocably removed upon the granting ofa patent disclosing them.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method for inhibiting tumor cell proliferation, comprisingcontacting a tumor cell with an effective amount of an isolatedantibody, or antigen-binding fragment thereof, wherein the antibody isselected from the group consisting of: (i) monoclonal antibodyDMF10.62.3 produced by hybridoma cell line ATCC No. PTA-377; (ii) achimeric version of antibody DMF10.62.3 comprising the light and heavychain variable regions of monoclonal antibody DMF10.62.3 and human lightand heavy chain constant regions, wherein said chimeric versionspecifically binds to the antigen to which monoclonal antibodyDMF10.62.3 binds; and (iii) a humanized version of antibody DMF10.62.3comprising the complementarity determining regions (CDRs) of monoclonalantibody DMF10.62.3, wherein said humanized version specifically bindsto the antigen to which monoclonal antibody DMF10.62.3 binds such thattumor cell proliferation is inhibited.
 2. The method according to claim1, wherein the antibody or antigen-binding fragment thereof induceshomotypic aggregation upon binding to the cell.
 3. The method accordingto claim 1, wherein the isolated antibody or antigen-binding fragmentinduces apoptosis in the cell.
 4. A method according to claim 1, whereinthe antibody or antigen-binding fragment thereof is selected from thegroup consisting of a thymic lymphoma cell, a T-cell lymphoma cell, aB-cell lymphoma cell, an osteosarcoma cell and an acute T-cell leukemiacell.
 5. A method according to claim 1, wherein the antibody orantigen-binding fragment thereof belongs to the class IgG.
 6. A methodaccording to claim 1, wherein the antibody or antigen-binding fragmentthereof is a composition comprising a pharmaceutically acceptablecarrier.
 7. A method according to claim 1, wherein the antibody orantigen-binding fragment thereof is conjugated to a moiety.
 8. A methodaccording to claim 1, wherein the moiety is selected from the groupconsisting of a radioactive molecule, a radionuclide, a sensitizermolecule, an imaging reagent, a radioisotope, a toxin, a cytotoxin, ananti-tumor agent, a chemotherapeutic agent, a modulator of the immunesystem, a cytokine and a reporter group.
 9. A method according to claim7, wherein the moiety is radionuclide selected from the group consistingof boron 10, ²¹¹At, ²¹²Pb, ²¹²Bi, ¹²⁵II, ¹³¹I, ³⁵S and ³H.
 10. A methodaccording to claim 7 wherein the moiety is a radionuclide and whereinthe radionuclide emits a particle selected from the group consisting ofan alpha particle, a beta particle, and a gamma particle.
 11. A methodaccording to claim 1, wherein the antibody or antigen-binding fragmentthereof comprises a detectable label.
 12. A method of selectivelydelivering a moiety to a tumor cell in a subject, the method comprisingadministering to the mammal an isolated antibody, or antigen-bindingfragment thereof, wherein the antibody is selected from the groupconsisting of: (i) monoclonal antibody DMF10.62.3 produced by hybridomacell line ATCC No. PTA-377; (ii) a chimeric version of antibodyDMF10.62.3 comprising the light and heavy chain variable regions ofmonoclonal antibody DMF10.62.3 and human light and heavy chain constantregions, wherein said chimeric version specifically binds to the antigento which monoclonal antibody DMF10.62.3 binds; and (iii) a humanizedversion of antibody DMF10.62.3 comprising the complementaritydetermining regions (CDRs) of monoclonal antibody DMF10.62.3, whereinsaid humanized version specifically binds to the antigen to whichmonoclonal antibody DMF10.62.3 binds, wherein the antibody is conjugatedto a moiety selected from the group consisting of a radioactivemolecule, a radionuclide, a sensitizer molecule, an imaging reagent, aradioisotope, a toxin, a cytotoxin, an anti-tumor agent, achemotherapeutic agent, a modulator of the immune system, a cytokine anda reporter group; and allowing sufficient time for the complex to bindto the tumor cell, such that the moiety is selectively delivered to thetumor cell in the mammal.
 13. A method according to claim 12, whereinthe moiety is radionuclide selected from the group consisting of boron10, ²¹¹At, ²¹²Pb, ²¹²Bi, ¹²⁵I, ¹³¹I, ³⁵S and ³H.
 14. A method accordingto claim 12 wherein the moiety is a radionuclide and wherein theradionuclide emits a particle selected from the group consisting of analpha particle, a beta particle, and a gamma particle.
 15. A method fordetecting a tumor cell in a subject, the method comprising contacting acell in a subject with an isolated antibody, or antigen-binding fragmentthereof, wherein the antibody is selected from the group consisting of:(i) monoclonal antibody DMF10.62.3 produced by hybridoma cell line ATCCNo. PTA-377; (ii) a chimeric version of antibody DMF10.62.3 comprisingthe light and heavy chain variable regions of monoclonal antibodyDMF10.62.3 and human light and heavy chain constant regions, whereinsaid chimeric version specifically binds to the antigen to whichmonoclonal antibody DMF10.62.3 binds; and (iii) a humanized version ofantibody DMF10.62.3 comprising the complementarity determining regions(CDRs) of monoclonal antibody DMF10.62.3, wherein said humanized versionspecifically binds to the antigen to which monoclonal antibodyDMF10.62.3 binds, under conditions that enable specific binding; anddetecting any specific binding of the antibody to the cell, whereinbinding indicates the presence of a tumor cell in the subject.
 16. Amethod according to claim 15, wherein the antibody or antigen-bindingfragment thereof comprises a detectable label.
 17. A method according toclaim 16, wherein the detectable label is selected from the groupconsisting of an enzyme, a prosthetic group, a fluorescent material, aluminescent material, a bioluminescent material, an electron denselabel, an MRI label, and a radioactive material.
 18. A method fordetecting a tumor cell in a subject, the method comprising contacting acell removed from the subject with an isolated antibody, orantigen-binding fragment thereof, wherein the antibody is selected fromthe group consisting of: (i) monoclonal antibody DMF10.62.3 produced byhybridoma cell line ATCC No. PTA-377; (ii) a chimeric version ofantibody DMF10.62.3 comprising the light and heavy chain variableregions of monoclonal antibody DMF10.62.3 and human light and heavychain constant regions, wherein said chimeric version specifically bindsto the antigen to which monoclonal antibody DMF10.62.3 binds; and (iii)a humanized version of antibody DMF10.62.3 comprising thecomplementarity determining regions (CDRs) of monoclonal antibodyDMF10.62.3, wherein said humanized version specifically binds to theantigen to which monoclonal antibody DMF10.62.3 binds, under conditionsthat enable specific binding; and detecting any specific binding of theantibody to the cell, wherein binding indicates the presence of a tumorcell in the subject.
 19. A method according to claim 18, wherein theantibody or antigen-binding fragment thereof comprises a detectablelabel.
 20. A method according to claim 19, wherein the detectable labelis selected from the group consisting of an enzyme, a prosthetic group,a fluorescent material, a luminescent material, a bioluminescentmaterial, an electron dense label, an MRI label, and a radioactivematerial.